Ramoplanin is a naturally-occurring glycosylated lipodepsipeptide antibiotic active against Gram-positive aerobic and anaerobic bacteria. Ramoplanin kills Gram-positive bacteria by inhibiting one of the enzymes needed to construct the bacterial cell wall. Ramoplanin was first described as antibiotic A/16686 produced by fermentation of Actinoplanes sp. ATCC 33076, as described in U.S. Pat. No. 4,303,646. It was subsequently found that three closely related components could be isolated from antibiotic A/16686, which components were named antibiotic A/16686 factors A1, A2, and A3 (Ciabatti et al., 1989, J. Antibiot (Tokyo), Vol. 42, No. 2, pp. 254-267). These substances as well as their preparation and uses are described in U.S. Pat. No. 4,427,656. Three additional factors designated A′1, A′2, and A′3 were later shown to be present in the fermentation medium and were shown to differ from the respective parent components of the original complex by lacking one mannose unit from the glycosidic group (Gastaldo et al., 1992, J. Ind. Microbiol. Vol. 11, No.1, pp.13-18).
Ramoplanin consists of a mixture of three related polypeptides having a common cyclic depsipeptide core structure on which is carried a dimannosyl glycosidic group. The three forms of ramoplanin are differentiated by the presence of various acylamide moieties derived from 8-, 9-, or 10-carbon fatty acids that decorate the glycosylated depsipeptide core structure.
Depsipeptides are cyclic or branched peptides containing an ester linkage between a carboxylate group of the peptide and a terminal or side-chain hydroxyl group of the peptide. The ramoplanin depsipeptide core structure contains 17 amino acids. The order of amino acids, from N-terminal to C-terminal, is as follows: amino acid 1: asparagine (Asn); amino acid 2: beta-hydroxyasparagine (HAsn); amino acid 3: 4-hydroxyphenylglycine (HPG); amino acid 4: ornithine (Orn); amino acid 5: threonine (Thr); amino acid 6: HPG; amino acid 7: HPG; amino acid 8: Thr; amino acid 9: phenylalanine (Phe); amino acid 10: Orn; amino acid 11: HPG; amino acid 12: Thr; amino acid 13: HPG; amino acid 14: glycine (Gly); amino acid 15: leucine (Leu); amino acid 16: alanine (Ala); amino acid 17: 3-chloro-4-hydroxyphenylglycine (CHPG). The peptide is cyclized by ester bond formation between the carboxylate group of the C-terminal CHPG and the hydroxyl group of HAsn. The N-terminus of Asn in position 1 is acylated by three different fatty acids, resulting in the three different components A1-A3. Two D-mannose sugars are attached to the HPG in position 11 by a hemiacetal bond.
Many low molecular weight peptides produced by bacteria are synthesized nonribosomally on large multifunctional proteins termed peptide synthetases. (Konz & Marahiel, 1999, Chem. Biol., Vol. 6, pp. R39-R48). Peptide synthetases contain repeated units that each recognize specific amino acids and catalyze their stepwise joining into a peptide chain. The identity of the amino acid recognized by a particular unit can be determined by comparison with other units of known specificity. In many peptide synthetases, there is a strict correlation between the order of repeated units in a peptide synthetase and the order in which the respective amino acids appear in the peptide product, making it possible to correlate peptides of known structure with putative genes encoding their synthesis, as demonstrated by the identification of the mycobactin biosynthetic gene cluster from the genome of Mycobacterium tuberculosis (Quadri et al., 1998, Chem. Biol. Vol. 5, pp. 631-645).
The repeating units of a peptide synthetase are composed of smaller units or “domains” that each carry out a specific role in the recognition, activation, modification and joining of amino acid precursors to form the peptide product. One type of domain, the adenylation (A) domain, is responsible for selectively recognizing and activating the amino acid that is to be incorporated by a particular unit of the peptide synthetase. The activated amino acid is joined to the peptide synthetase through another type of domain, the thiolation (T) domain, that is generally located adjacent to the A domain. Amino acids joined to successive units of the peptide synthetase are subsequently linked together by the formation of amide bonds catalyzed by another type of domain, the condensation (C) domain.
Although the structure of ramoplanin has been identified, there remains the need to obtain novel structures with new activities or enhanced properties. There is also a need to improve production of ramoplanin. Accordingly, there is a need for genetic information regarding the biosynthesis of ramoplanin.